Autonomous sensing molecules (asm)

Abstract

Provided herein is a sensing molecule comprising an aptamer, a linker, and a DNAzyme, wherein the DNAzyme is inactive in the absence of a target and active in the presence of a target, and wherein the linker is covalently bound to the aptamer and the DNAzyme. Such sensing molecules may be used for detecting a target molecule in a sample or for use in diagnosis of a disease or disorder.

1 . A sensing molecule comprising an aptamer, a linker, and a DNAzyme, wherein the DNAzyme is covalently bound to the aptamer via the linker, wherein in the absence of a target the DNAzyme is associated with the aptamer and is inactive, and wherein in the presence of a target the DNAzyme is active. 
     
     
         2 . The sensing molecule of  claim 1 , wherein the aptamer and the DNAzyme are directly associated, preferably via hydrogen bonds. 
     
     
         3 . The sensing molecule of  claim 1 , wherein the DNAzyme folds into an enzymatically active structure upon binding of the aptamer to the target. 
     
     
         4 . The sensing molecule of  claim 1 , wherein the DNAzyme mimicks a reporter enzyme, preferably an alkaline phosphatase (AP), a horseradish peroxidase (HRP), a luciferase, or a glucose oxidase (GOx). 
     
     
         5 . The sensing molecule of  claim 1 , wherein the DNAzyme is a HRP-mimicking enzyme, preferably a hemin-binding HRP-mimicking G-quadruplex DNAzyme. 
     
     
         6 . The sensing molecule of  claim 1 , wherein the aptamer is partially reverse complementary to the DNAzyme. 
     
     
         7 . The sensing molecule of  claim 1 , wherein the aptamer is a DNA aptamer or a RNA aptamer. 
     
     
         8 . The sensing molecule of  claim 1 , wherein the aptamer comprises at least 25 nucleotides, preferable at least 50 nucleotides, more preferable 25 to 75 nucleotides and/or nucleotide analogs. 
     
     
         9 . The sensing molecule of  claim 1 , wherein the aptamer comprises at least one chemically modified nucleotide or nucleotide analog, preferably wherein any one of at least 40%, 50% or 60% of the nucleotides and/or nucleotide analogs are chemically modified. 
     
     
         10 . The sensing molecule of  claim 1 , wherein the aptamer comprises at least one nucleotide and/or nucleotide analog with one or more covalently attached amino acid residue(s) and/or one or more amino acid analog(s), preferably wherein one or two amino acid or amino acid analog residue(s) are covalently attached to the nucleotide and/or nucleotide analog. 
     
     
         11 . The sensing molecule of  claim 1 , wherein the linker comprises at least 3 nucleotides and/or nucleotide analogs, preferably 3 to 100 nucleotides and/or nucleotide analogs. 
     
     
         12 . The sensing molecule of  claim 1 , wherein the linker enables association of the aptamer and the DNAzyme in the absence of a target, preferably wherein the linker comprises a self-complementary sequence. 
     
     
         13 . The sensing molecule of  claim 1 , wherein the sensing molecule is a single-stranded DNA molecule. 
     
     
         14 . The sensing molecule of  claim 1 , wherein the target is selected from the group consisting of metal ions, small organic molecules, organic dyes, peptides, proteins, surface proteins, lipopolysaccharides, whole cells and microorganisms. 
     
     
         15 . A method for detecting a target molecule in a sample, comprising:
 (i) contacting the sensing molecule of  claim 1  with the sample;   (ii) determining the enzymatic activity of the DNAzyme; and   (iii) detecting the target molecule, wherein enzymatic activity is indicative of the presence of the target.   
     
     
         16 . The method of  claim 15 , wherein the enzymatic activity is determined by a colorimetric, fluorescent or electrochemical signal. 
     
     
         17 . The method of  claim 15 , wherein the sample is an industrial or environmental sample, preferably a sample from soil, water, or waste-streams; or a biological sample, preferably from body fluids, swabs or tissues. 
     
     
         18 . (canceled) 
     
     
         19 . The sensing molecule of  claim 1 , wherein the aptamer and the DNAzyme are indirectly associated via helper oligonucleotides.